Mycopathologia vol. 55, 3, pag. 179-183, 1975

PENETRATION OF TRICHOPHYTON TERRESTRE IN H U M A N HAIR

Yuan C. HSU & Paul A. VOLZ Department of Biology, Eastern Michigan University Ypsilanti, Michigan 48197 USA

& Taplin, 1970). The lack of pathogenicity of this dermatophyte was a factor in species selection for exomycological studies on the NASA Apollo program (Volz et al., 1974). The medically designed studies directed attention to survival and change of nonpathogenic microorganisms when subjected to various spaceflight parameters (Taylor, 1970). Variation in survival and other factors were noted in preliminary investigations of the postflight selected fungal phenotypes (Volz, 1973). The penetration and progressive development of T. terrestre wild type in human hair was examined with light and electron microscopy. Investigations are underway that demonstrate variation in penetrability of retrieved T. terrestre phenotypes after exposure to specific parameters of space during the flight of Apollo 16 (Veselenak & Volz, 1972).

(BBL) in petri plate~ then inoculated with conidia and hyphal fragments grown on Sabouraud maltose agar (Difco). The inoculum was centrally placed on the agar hair plates and colony growth of T. terrestre radiated from the point of inoculation. Mycelial growth was supported by the hair while moisture was provided by the nutrient fi'ee purified agar. Cultures were allowed to incubate 2~4 weeks before the infected hair was removed and examined microscopically. Hairs retained for light microscopy were slide mounted in lactophenol cotton blue. Infected hairs selected for electron microscopy were fixed in 0.05 M phosphate buffered 3 o/~,glutaraldehyde3 % acrolein at pH 6.8 after growth periods of 10, 20, and 30 days. After 2 hours of fixation the infected hair was repeatedly washed in the 0.05 M phosphate buffer and post-fixed in 0.05 M phosphate buffered osmium tetroxide for one hour. The material was then washed several times in distilled H20 followed by a dehydration series of ETOH to propylene oxide. The epon araldite combination by Anderson & Ellis (1965) was selected for embedding and the material was polymerized at 60 ~ on a flat silicone rubber mold. After polymerization the embedded hair was removed from the mold, oriented and glued with the epon mixture to a blank specimen block for sectioning on a Sorvall Porter-Blum ultramicrotome. The sectioned hair was collected on formvar supported 100 mesh copper grids and stained with Reynolds' lead citrate (Reynolds, 1963). Specimen examination was made with a Zeiss EM9-S-2 electron microscope.

Materials and methods

Results

One collection of sterilized hair from one subject was placed aseptically on the surface of 4% purified agar

Ectothrix mycelial growth of T. terrestre is apparent as soon as colony formation occurs (fig. 4). Colony growth increases in density on the hair with an increase in colony age. With dense ectothrix growth well established, forma-

Abstract

The systematic human hair degradation by Trichophyton terrestre was examined by electron and light microscopy. The cuticular and hair shaft regions were readily decomposed by the wild type or parent strain selected for phenotype studies after exposure to spaceflight parameters.

Introduction

The in vitro and the absence of in vivo growth of Trichophyton terrestre on human hair is well established (Rebell

Received : 4.X. 1974.

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Figs. t-9. Light microscopy of 1. Non-infected normal human hair, x 1,275, 2. Initial invagination and raised cuticle of hair infected with T. terrestre, 20 days growth, x 1,000, 3. Decomposed infected hair with deep invagination, cuticle absent, at 30 days incubation, x 1,000, 4. Aerial mycelial growth of T. terrestre on hair shaft, 14 days growth, x 95 Electron microscopy of 5. Infected hair showing cuticle and hair shaft impregnated with T. terrestre hyphae, prominent melinin pigments, 20 days growth, x 2,700, 6. Initial infection in cuticle, cuticle layer adjacent to hyphae is partially digested, 10 days growth, x 6,900, 7. Initial infection in hair interior with young actively growing hyphae, 20 days growth, x 14,250, 8. Decomposition of central hair region, 25 days growth, x 14,250, 9. Highly branched mature hyphal system in completely digested central region of hair, 30 days growth, x 6,900.

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tion of invaginations in the hair shaft is more commonly found. Soon the hyphal elements of the fungus are equally developed both on the hair shaft surface and within the hair. Normal hair in light microscopy demonstrates little density variation even after autoclaving for inoculation of the keratinophilic fungus (fig. 1). The cuticle soon enlarges with the growth of mycelium, and invaginations form on the hair surface and develop inward into the hair shaft (fig. 2). With dense hyphal growth on the hair surface the cuticle soon disappears and the hair interior gradually is digested. Inward invaginations are commonly found and ultimately dissect the entire hair diameter (fig. 3). The normal hair structure disappears as deterioration of the hair becomes complete.

A cross section electron micrograph reveals hyphal growth channels within the hair (fig. 5). Random cuticular layers of hyphae and surface growth are less organized than internal hyphal development within the hair shaft. Melanin granules in the hair (Montagna & Ellis, 1958) are visible as electron dense structures. The granules are not damaged by the advancing mycelium. On close examination of infected hair regions, hyphae are first established and associated with the cuticle. Cuticular layers adjacent to advancing hyphae soon demonstrate the activity of extracellular digestion at 10 days incubation (fig. 6). Hyphal filaments gradually penetrate the hair interior (fig. 7). Soon the mycelial channels within the hair are common. Hair structure disappears as hair digestion progresses and hyphal channels merge (fig. 8). A t completion of hair disintegration a highly branched my-

celial network remains (fig. 9). Melanin granules remain undisturbed in the presence of mature mycelium while the surrounding hair structure is completely digested. Hair destruction is quite thorough after 30 days of T. terrestre growth. However, variation in the rate of hair deterioration does occur according to the individual hair source and involved fungal strain.

Discussion

The in vitro invasion of Trichophyton terrestre into human hair systematically destroys the hair shaft. Aerial growth from the hair becomes visible to the unaided eye as colony growth progresses. Initial invasion occurs in the cuticle then hyphal filaments invade the central or fibrous keratin 181

portion of the hair forming channels within the hair. Mycelium in the hair is highly branched while aerial hyphal filaments branch very rarely. As hyphal growth continues, cuticular layers or overlapping scales adjacent to the branching hyphae deteriorate and channels in the hair gradually increase in size as the hair is slowly digested. The melanin granules in the hair appear to be the only portion of the hair demonstrating immunity against hair decomposition imposed by the advancing mycelium ofT. terrestre. A recent study in electron microscopy revealed an increase in size and number of melanosome granules in skin infected with Malasseziafurfur (Charles et al., 1973). The cuticle layers or scales are firmly bonded together forming a protective outer layer to the hair cortex which is composed of fibrous keratin in a whorl design or pattern. The fibrous keratin is enclosed in modified cell membranes and nonkeratinous residues (Mercer & Verma, 1963). Trichophyton mentagrophytes readily attacks the separating membranes and residues, and in addition Mercer & Verma (1963) also observed the enzymatic disintegration of the cortical keratin. The penetrating hyphae of T. terrestre gain entrance to the inner portion of the hair following the separation of membraneous tracts of cortical keratin. Channels of hyphal penetration soon appear within the hair as enzymatic digestion deteriorates the hair structure. The mechanism of hair penetration and destruction by T. mentagrophytes, a virulent dermatophyte, is similar to that found in the nonpathogen T. terrestre. However, electron dense pigments are not affected by the extracellular enzymes of the nonpathogen. Scanning electron microscopy was utilized for surface observation of Malassezia furfur on hair (Barnes et al., 1973). Direct involvement of both hyphal and spheroidal cell types on the hair shaft was seen. However, no obvious evidence of tissue pathology on the hair surface was noticed. Other SEM studies on hair shafts included patients with psoriasis. The psoriatic plaques have associated hairs with significantly thinner shafts and obvious dystrophic changes of the hair cuticle (Orfanos et al., 1970: Wyatt et al., 1972). The invading hyphae of Candida albicans is intracellular in dermal tissue with penetration directly through membrane systems to the host cell cytoplasm when examined with the electron microscope (Cawson & Rajasingham, 1972). Trichophyton terrestre causes complete destruction of hair without opportunistic penetration into healthy mature cells. Hair invasion also appears to be one habitat limit of Trichosporon eutaneum in vivo and colonization of hairs devoid of additional supplemental nutrients allows for further experimentation (Smith, 182

Murtishaw & McBride, 1973). The technique of using sterile hair in hair baiting soil samples is a well known method for isolation of keratinophilic fungi. The test species under study proved to be a good selection for experiments in hair deterioration with the restrictive use of a nonpathogen to reduce possible human involvement in a manned spaceflight.

Acknowledgments Support for this study as part of the Apollo MEED Project was kindly received by P.A.V. from the National Research Council and the National Aeronautics and Space Administration. Appreciation is extended to Dr. R. A. Giles for his support in the MEED Project sections housed at Eastern Michigan University, Ypsilanti, and to D. E. Jerger and J. M. Veselenak for technical assistance at the Lunar Receiving Laboratory NASA JSC, Houston.

References Anderson, W. A. & R. A. Ellis. 1965. Ultrastructure of Trypanosoma lewsi; flagellum, microtubules, and the kinetoplast. J. Protozool. 12: 483499. Barnes, W. G., G. C. Sauer & J. D. Arnold. 1973. Scanning electron microscopy of tinea versicolor organisms. Arch. Dermatol. 107: 392-394. Cawson, R. A. & K. C. Rajasingham. 1972. Ultrastructural features of the invasive phase of Candida albicans. Br. J. Dermatol. 85: 435443. Charles, C. R., D. L. Sire, B. L. Johnson & J. G. Beidler. 1973. Hypopigmentation in tinea versicolor: A histochemical and electron microscopic study. Int. J. Dermatol. 12: 48-58. Mercer, E. H. & B. S. Verma. 1963. Hair digested by Trichophyton mentagrophytes. Archives of Dermatol. 87: 357-360. Montagna, W. & R. A. Ellis. 1958. The Biologyof Hair Growth. Academic Press, New York. 520 pp. Orfanos, C., G. Mahrle & R. Christenhusz. 1970. Verhornungsst6rungen am Haar bei Psoriasis. Eine Studie im RasterElektronenmikroskop. Arch. Klin. Exp. Dermatol. 246: 107-114. Rebell, G. & D. Taplin. 1970. Dermatophytes, Their Recognition and Identification, Revised Edition. University of Miami Press, Coral Gables, Florida. 124 pp. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electropaque stain in electron microscopy. J. Cell Biol. 17: 208 213. Smith, J. D., W. A. Murtishaw & M. E. McBride. 1973. White piedra (Trichosporosis). Arch. Dermatol. 107: 4394-442. Taylor, A. M. 1970. Design feasibility study for construction of a microbial ecology evaluation device (MEED). Aerojet Medical and Biological Systems. E1 Monte, California NAS 9-10820.

Veselenak, J. M. & P. A. Volz. 1972. The Apollo 16 MEED mycology incorporated tests. In: The ASEE 1972 Annual Publication, Washington, D.C. Volz, P. A. 1973. Mycological studies housed in the Apollo 16 microbial ecology evaluation device. In: NASA TMX Proceedings of the Microbial Response to Space Environment Symposium. p. 121 125. Volz, P. A., Y. C. Hsu, J. L. Hiser, J. M. Veselenak & D. E. Jerger. 1974. The microbial ecology evaluation device mycology spaceflight studies of Apollo 16. Mycopath. Mycol. Appl. 54:221 233. Wyatt, E., E. Bottoms & S. Comaish. 1972. Abnormal hair shafts in psoriasis on scanning electron microscopy. Br. J. Dermatol. 87: 368-373.

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Penetration of Trichophyton terrestre in human hair.

The systematic human hair degradation by Trichophyton terrestre was examined by electron and light microscopy. The cuticular and hair shaft regions we...
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